Alternative splicing (AS) is an important regulatory mechanism in higher eukaryotes (P. A. Sharp, Cell 77, 805-8152 (1994). It is thought to be one of the important mechanisms for differential expression related to tissue or development stage specificity. It is known to play a major role in numerous biological systems, including human antibody responses, sex determination in Drosophila, and (S. Stamm, M. Q. Zhang, T. G. Marr and D. M. Helfman, Nucleic Acids Research 22, 1515-1526 (1994); B. Chabot, Trends Genet. 12, 472-478 (1996); R. E. Breitbart, A. Andreadis, B. Nadal-Ginard, Annual Rev. Biochem., 56, 467-495 (1987); C. W. Smith, J. G. Patton, B. Nadal-Ginard, Annu. Rev. Genet., 27, 527-577 (1989).
Until recently it was commonly believed that alternative splicing existed in only a small fraction of genes (about 5%). A recent observation based on literature survey of known genes revises this estimate to as high as stating that at least 30% of human genes are alternatively spliced (M. S. Gelfand, I. Dubchak, I. Draluk and M. Zorn, Nucleic Acids Research 27, 301-302 (1999). The importance of the actual frequency of this phenomenon lies not only in the direct impact on the number of proteins created (100,000 human genes, for example, would be translated to a much higher number of proteins), but also in the diversity of functionality derived from the process.
Several mechanisms at different stages may be held responsible for the complexity of higher eukaryote which include: alternative splicing at the transcription level, RNA editing at the post-transcriptional level, and post-translational modifications are the ones characterized to date.
Kinases are enzymes that catalyze the phosphorylation of target proteins. This phosphorylation event causes activation, or at times inactivation, of the target protein. Kinases are divided into two major groups, based on the amino acid residue that they phosphorylate: tyrosine kinases and the more abundant group, serine/threonine kinases play a key role in the signal transduction mechanisms that control diverse biological processes, including cellular proliferation, differentiation, adhesion, mobility, survival and apoptosis, the immune response, neutrotransmission and cellular metabolism.
Alterations in the activity of various kinases have been extensively studied and linked to the pathogenesis of most major diseases, including cancer, central nervous system disorders, immune diseases/inflammations, asthma, autoimmune disease, arthritis, graft vs. host disease and transplantation complications, cardiovascular disease, liver disease, hormonal and metabolic disorders, osteoporosis, AIDS and other infectious disease.
A dominant negative is an inactive form of a protein that reduces or eliminates the activity of its active form. It may act by binding to the active protein and rendering it inactive, or where it is an enzyme by binding the target protein without enzymatically activating the protein, thus blocking and preventing the active enzymes from binding and activating the target protein. Dominant negative kinases might be alternatively spliced gene products, which lost an important site or domain, and thus became enzymatically inactive and therefor act as inhibitors of the active kinases, for example, by binding to the target protein without phosphorylating it and blocking the binding to other active kinases. By inhibiting the activity of the active kinases, the dominant negative kinases may interfere with a disease related process, such as cell proliferation in a tumor Kinase variants can act as dominant-negative inhibitors through a variety of mechanisms depending on their lost or defected site or domain. For example, truncated Growth Hormone Receptor that lacks most of its intracellular domain has been shown to heterodimerize with the full-length receptor, thus causing inhibition of signaling by Growth Hormone (Ross, R. J. M., Growth hormone & IGF Research, 9:42-46, 1999).
Direct specific inhibition of Protein Kinase C (PKC), for example, has been shown to initiate apoptosis in a variety of malignant cell types. Recently, two new alternatively spliced forms of PKC delta has been reported. The first—PKC delta II (mouse)—has 73 bp (26 amino acid) insertion at the caspase recognition region turning it to a caspase-resistant form.
The second—PKC delta III (rat)—has 83 bp insertion in the same region causing frame shift and forming truncated protein without the catalytic domain. That truncated form has been considered to act in a dominant-negative manner against the intact sub-type (Ueyama, T., et al., Biochem. Biophys. Research Communications, 269:557-563, 2000).
Many kinase inhibitors have clinical implications. They serve as drug targets or show potential for the treatment of many diseases, such as potential in the treatment of certain cancers and treatment of type II diabetes. Tyrosine kinase inhibitors have shown various effects in treatment of neurodegenerative disease including Alzheimer's and Parkinson's disease, prevention of restenosis following angioplasty, treatment of atherosclerosis, inflammation, thrombosis, autoimmune disease, allergy, asthma, transplant rejection, psoriasis, fibrosis, dwarfism and other growth disorders, as well as inhibiting the proliferation and function of natural killer cells (immunosuppressant effect).
Protein Kinase C (PKC) inhibitors are used in cardiovascular disease, diabetes, CNS disorders, arthritis, septic shock and inflammation bowel disease, and PKC inhibitors have shown also potential in the treatment of certain cancers and have been shown to initiate apoptosis in a variety of malignant cell types. p38 Kinase inhibitors may be effective in attenuating COX-2-mediated prostaglandins, inflammation, treatment of rheumatoid arthritis and inflammatory bowel diseases.